The present invention relates to polymers which are useful as supports in solid phase organic synthesis (SPOS).
Cross-linked insoluble polystyrene resin supports, normally in the form of spherical beads, are one of the most important types of substrate used for SPOS. They are relatively cheap and robust, can be made with a high functional group loading, with a wide range of functionalities available, and they swell in a variety of non hydroxylic solvents, thus giving access to a variety of reagents. Their main drawbacks are the lack of swelling in water or alcohols, and the close proximity of the functional groups to the hydrophobic core, which can cause steric hindrance to reactions, slowing or even preventing them, and may also give rise to poor NMR spectra due to slow relaxation times.
xe2x80x98Tentagelxe2x80x99(trademark) (trademark of Rapp Polymere GMBH) polymer supports are used widely in SPOS. These polymer supports have a polystyrene core with polyethylene glycol (MWt ca 4000) chains grafted onto this core. The terminal hydroxy groups are then functionalised to allow the synthesis to take place. The polymer supports are useful in that they are hydrophilic and so can swell in polar solvents such as water and alcohols as well as the usual solvents (toluene, tetrahydrofuran, dichloromethane) used for SPOS. The functional groups are well separated from the crosslinked polystyrene core and are thus unhindered for reaction and very mobile for good NMR analysis. The main deficiencies are that the functional group loading is low and they possess an acid labile benzyl ether linkage, thus restricting the range of reaction conditions under which they can be employed.
We have designed and made a series of novel polymer resin supports that can have a higher functional loading than that of xe2x80x98Tentagelxe2x80x99(trademark) and yet, in certain embodiments, still swell in water or alcohols and at the same time do not have unduly slow relaxation times enabling NMR to be used.
Accordingly the present invention provides a polymer support which comprises hydroxypolyC2-4 alkyleneoxy chains attached to a cross-linked polymer wherein the hydroxypolyC2-4 alkyleneoxy chain contains from 2 to 8 C2-4 alkyleneoxy groups and wherein the resulting polymer support has from about 0.1 to about 5 meq free hydroxy groups per gram of polymer.
The hydroxypolyC2-4alkyleneoxy chains in the supports according to the present invention are often selected from hydroxypolyethyleneoxy (HO(CH2CH2O)2-8xe2x80x94), hydroxypolypropyleneoxy (HO(CH2CH(CH3)O)2-8xe2x80x94) and hydroxypolybutyleneoxy (HO(CH2CH(C2H5)O)2-8xe2x80x94) chains. In a preferred embodiment of the invention the hydroxypolyC2-4alkyleneoxy chain is hydroxypolyethyleneoxy.
The number of C2-4alkyleneoxy groups in the hydroxypolyC2-4alkyleneoxy chain can range from 2 to 8, but is preferably from 3 to 5. Most preferably, there are 4 C2-4alkyleneoxy groups in the hydroxypolyC2-4alkyleneoxy chain.
In the most preferred embodiment of the invention the hydroxypolyC2-4alkyleneoxy chain is hydroxytetraethyleneoxy (HO(CH2CH2O)4xe2x80x94).
The cross-linked polymer in the supports according to the present invention may be, for example, a cross-linked polymer obtainable by polymerising a monomer mixture comprising at least one monomer selected from hydroxystyrene, hydroxymethylstyrene, chloromethylstyrene, hydroxyethyl (meth)acrylate, hydroxypropyl (meth)acrylate and N-methylol (meth)acrylamide; wherein the phenyl ring in the styrenes is optionally substituted by 1 or 2 substituents often selected from methyl, ethyl, propyl, fluoro, chloro and bromo and wherein hydroxy groups, especially phenolic hydroxy groups, which may be present in the monomers are optionally protected and may subsequently be deprotected.
Preferably the cross-linked polymer is a copolymer comprising phenylethylene and hydroxyphenylethylene units or phenylethylene and chloromethylphenylethylene units, and more preferably a copolymer comprising phenylethylene and 4-hydroxyphenylethylene units or phenylethylene and 4-chloromethylphenylethylene units. The polymer support formed from a cross-linked polymer comprising phenylethylene and 4-hydroxyphenylethylene units has the additional advantage of not containing an acid labile benzyl ether linkage. Even more preferably the cross-linked polymer is a copolymer comprising phenylethylene and 4-hydroxyphenylethylene units. The cross-linked polymer can often be derived by polymerising a monomer mixture comprising styrene and optionally protected hydroxystyrene and subsequently deprotecting the hydroxy group if protected, or a monomer mixture comprising styrene and chloromethylstyrene. Most preferably the cross-linked copolymer is obtainable by polymerising styrene and optionally protected 4-hydroxystyrene under conditions to produce cross-linking, and subsequently deprotecting the hydroxy group if protected.
Protecting groups for hydroxy groups may in general be chosen from any of the groups described in the literature or known to the skilled chemist as appropriate for the protection of the hydroxy group in question, and may be introduced by conventional methods. Where protecting groups are present during a polymerisation process, the protecting groups are selected so as to survive the conditions of the polymerisation.
Protecting groups may be removed by any convenient method as described in the literature or known to the skilled chemist as appropriate for the removal of the protecting group with minimum disturbance of groups elsewhere in the molecule.
Specific examples of protecting groups are given below for the sake of convenience, in which xe2x80x9clowerxe2x80x9d signifies that the group to which it is applied preferably has 1-4 carbon atoms. It will be understood that these examples are not exhaustive. Where specific examples of methods for the removal of protecting groups are given below these are similarly not exhaustive. The use of protecting groups and methods of deprotection not specifically mentioned is of course within the scope of the invention.
Examples of hydroxy protecting groups include tetrahydropyranyl, lower alkyl groups (for example t-butyl), lower alkenyl groups (for example allyl); lower alkanoyl groups (for example acetyl); lower alkoxycarbonyl groups (for example t-butoxycarbonyl); lower alkenyloxycarbonyl groups (for example allyloxycarbonyl); phenyl lower alkoxycarbonyl groups (for example benzyloxycarbonyl, p-methoxybenzyloxycarbonyl, o-nitrobenzyloxy-carbonyl, p-nitrobenzyloxycarbonyl); tri lower alkysilyl (for example trimethylsilyl, t-butyldimethylsilyl) and phenyl lower alkyl (for example benzyl) groups.
The hydroxy protecting group may subsequently be removed to give the cross-linked polymer containing free hydroxy groups.
Methods appropriate for removal of hydroxy protecting groups include, for example, acid-, base-, metal- or enzymically-catalysed hydrolysis, for groups such as p-nitrobenzyloxycarbonyl, hydrogenation and for groups aso-nitrobenzyloxycarbonyl, photolytically. The reader is referred to Advanced Organic Chemistry, 4th Edition, by Jerry March, published by John Wiley and Sons 1992, for general guidance on reaction conditions and reagents. The reader is referred to Protective Groups in Organic Synthesis, 2nd Edition, by Green et al., published by John Wiley and Sons for general guidance on protecting groups.
Acetyl is a preferred protecting group for the hydroxy group in protected-hydroxy styrene.
When the cross-linked polymer is produced by polymerisiation of a mixture of monomers comprising styrene and optionally protected hydroxystyrene or chloromethylstyrene, the weight percentage of optionally protected-hydroxystyrene or chloromethylstyrene of the total weight of optionally protected-hydroxystyrene or chloromethylstyrene plus styrene is preferably in the range of from 1-99%, more preferably in the range of from 5-80% and most preferably from 15% to 70%.
The extent of cross linking in the polymers is determined by the concentration of cross linking monomer in the polymerisation reaction. Generally the weight % of cross-linking monomer is in the range of from 0.1 to 70%, commonly from 0.5 to 20%, such as from 1 to 10%, and most preferably no more than 5% by weight. Polymers comprising no more than 20% by weight of cross-linking monomer are generally swellable, whilst polymers comprising greater than 20% of crosslinking monomer are generally not swellable.
Suitable cross-linking monomers include divinyl benzene (DVB) or multifunctional (meth)acrylates such as di/tri acrylates or di/tri methacrylates such as ethylene glycol diacrylate, ethylene glycol dimethacrylate, trimethylopropane trimethacrylate, trivinylbenzene or N,Nxe2x80x2-bis-acryloyl ethylene diamine. Preferably the cross-linking monomer is DVB.
Preferably 0.5 to 5% by weight of DVB is used. Most preferably 1 to 3% by weight DVB is used.
The polymer support according to the present invention can be prepared by reacting an appropriately functionalised cross-linked polymer with a poly(C2-4alkyleneoxy)glycol or derivative thereof. Commonly, the appropriately functionalised cross-linked polymer comprises a free hydroxy group, which is reacted with a poly(C2-4alkyleneoxy)glycol derivative comprising a leaving group attached to one end of the poly(alkyleneoxy) chain, with the hydroxy group at the other end of the chain being protected by a hydroxy-protecting group. Alternatively, a cross-linked copolymer containing a free hydroxy group can be reacted with a mono-protected poly(alkyleneoxy)glycol.
Cross-linked polymers containing a free hydroxy group are usually produced as beads which range in size from 10 xcexcm to 2000 xcexcm. Preferably the bead size is from 50 xcexcm to 1000 xcexcm and most preferably from 75 xcexcm to 500 xcexcm. The cross-linked polymer beads are generally produced by an aqueous suspension polymerisation process, for example see Journal of Applied Polymer Science, 1982, 27, 133-138, incorporated herein by reference.
In aqueous suspension polymerisation, the monomers are suspended as droplets (1-1000 xcexcm) in water. Stabilisers are usually added to prevent agglomeration of the droplets, for example polyvinyl alcohol, polyacrylic acid, polyvinyl pyrrolidone, polyalkylene oxide, barium sulphate, magnesium sulphate or sodium sulphate. The suspension is also normally stirred to maintain the suspension.
A free radical initiator is preferably used to initiate polymerisation. The type of initiator will generally be selected based on the monomers used. Examples of preferred free radical initiators include benzoyl peroxide, dioctanoyl peroxide, 2,2xe2x80x2-azobisisobutyronitrile and 2,2xe2x80x2-azobis(2,4-dimethylvaleronitrile).
Polymerisation is typically assisted by heating the mixture in the range of 15xc2x0 C. to 160xc2x0 C., preferably 50xc2x0 C. to 90xc2x0 C. It will be recognised that the temperature to which the mixture can be heated depends upon the type of monomer and initiator employed.
The resultant polymer may then be washed with suitable solvents such as tetrahydrofuran, methanol and water, dried and bead size classified, for example, by sieving.
Suitable protecting groups for hydroxy are described above. A preferred protecting group for mono-protected poly(alkyleneoxy)glycols and for poly(C2-4alkyleneoxy)glycol derivatives comprising a leaving group attached to one end of the poly(alkyleneoxy) chain is tetrahydropyranyl.
Polymer supports according to the present invention can be prepared by the reaction between a cross-linked polymer comprising a free hydroxy group and a mono-protected poly(alkyleneoxy)glycol derivative wherein the free hydroxy group has been converted into a leaving group, such as a tosylate, mesylate or halo, such as chloro or bromo, group, in the presence of a strong base, such as sodium methoxide or ethoxide, or sodium hydride, using conditions known for the Williamson ether synthesis.
Cross-linked polymers which do not contain free hydroxy groups but contain groups which can be converted to hydroxy groups can be converted to cross-linked copolymers with free hydroxy groups before reacting with the mono-protected poly(alkyleneoxy)glycol. In some cases this may be carried out in situ.
A cross linked copolymer formed from styrene and chloromethylstyrene may be reacted with the mono-protected poly(alkyleneoxy)glycol in the presence of a strong base, such as sodium methoxide or ethoxide, or sodium hydride, using conditions known for the Williamson ether synthesis.
The polymer supports according to the present invention can also conveniently be prepared by reaction between a cross-linked polymer containing free hydroxy groups and a mono-protected poly(alkyleneoxy)glycol under conditions known for the Mitsunobu reaction. This typically involves reacting the reagents together in the presence of di(C1-4alkyl)azo-carboxylate or 1xe2x80x2,1xe2x80x2-(azodicarbonyl) dipiperidine and a phosphorous reagent such as tributylphosphine or triphenylphosphine in an inert solvent such as toluene, benzene, tetrahydrofuran (THF) or diethylether, at non-extreme temperatures such as in the range xe2x88x9220xc2x0 C. to ambient temperatures. (See Progress in the Mitsunobu Reaction. A Review, David L. Hughes, Organic Preparations and Procedures Int., 28 (2), 127-164 (1996)).
The polymer supports according to the present invention can alternatively, be prepared by polymerisation of a monomer comprising a hydroxy(polyC2-4alkyleneoxy) moiety, preferably in which the free hydroxy of the hydroxy(polyC2-4alkyleneoxy) moiety is protected with a suitable hydroxy protecting group, under conditions to produce cross-linking. In many such embodiments, a styrene monomer substituted on the phenyl moiety, preferably at the 4-position, by an optionally protected hydroxy(polyC2-4alkyleneoxy) moiety, is polymerised in the presence of a cross-linking monomer. For example, a polymer support which could be prepared by first making the copolymer derived by polymerising styrene, optionally protected 4-hydroxystyrene and appropriate cross-linking monomer and then reacting the cross-linked copolymer with the poly(alkyleneoxy)glycol can alternatively be prepared by polymerising styrene and 4-(optionally protected)-hydroxy(polyalkyleneoxy)styrene together with the appropriate cross-linking monomer. Cross-linking monomers and proportions employed can be as described above for the preparation of cross-linked polymers.
The invention, in its broadest aspect, relates to the particular polymer supports however prepared.
The polymer support according to the present invention has a hydroxy functionality of from 0.1 to about 5, for example up to 4.8 meq (milliequivalents) of hydroxy per gram of polymer, and often from 0.5 to 3.5, commonly 1.0 to 3.3 meq per gram for example from 1.5 to 3 meq per gram of polymer. In many embodiments, polymer supports having from 0.5 to 2 meq of hydroxy per gram of polymer can advantageously be employed.
In a preferred aspect of the invention, the polymer support comprises hydroxypolyethyleneoxy chains attached to a cross-linked copolymer via an ether linkage, wherein the cross-linked polymer can be formed by polymerising styrene and optionally-protected 4-hydroxystyrene, and wherein the hydroxypolyethyleneoxy chain contains from 2 to 8 ethyleneoxy groups and wherein resulting polymer support has about 0.1 to about 5 meq free hydroxy groups per gram of polymer.
In a most preferred aspect of the invention, the polymer support comprises hydroxytetraethyleneoxy chains attached to a cross-linked copolymer via an ether linkage, wherein the cross-linked polymer can be formed by polymerising styrene and 4-acetoxystyrene and subsequently removing the acetyl group, and wherein the resulting polymer support has about 0.1 to about 5 meq free hydroxy groups per gram of polymer.
Another aspect of the invention relates to a polymer support which comprises hydroxypolyC2-4 alkyleneoxy chains, wherein the terminal hydroxy group is protected, attached to a cross-linked polymer wherein the hydroxypolyC2-4 alkyleneoxy chain contains from 2 to 8 C2-4 alkyleneoxy groups.
Preferred protecting groups for the terminal hydroxy group in the hydroxypolyC2-4 alkyleneoxy chain are acetyl, benzyl, benzoyl, tri(alkyl)silyl and tetrahydropyranyl groups.
The hydroxy-protecting group can be removed using standard techniques known in the art. For example the tetrahydropyranyl group may be removed with p-toluene sulphonic acid.
Removal of the hydroxy-protecting group gives an unprotected polymer support of the present invention comprising hydroxypolyC2-4alkyleneoxy chains on a cross-linked polymer wherein the hydroxypolyC2-4alkyleneoxy chains contain from 2 to 8 oxyC2-4alkylene groups.
The polymer supports according to the present invention are of use in SPOS, and such use, wherein a process of solid phase organic synthesis occurs on a support according to the present invention, forms another aspect of the present invention. They preferably have a swell ratio of at least 4 in chloroform. They are preferably used in the form of spherical beads.
Traditional organic chemistry has usually been carried out in solution. Methods were developed for the synthesis of oligomers, such as peptides and nucleotides, on a solid phase (typically beads) (Merrifield: Adv Enzymol, 32, 221 (1969)), and this was then very amenable to automation. Ligands were attached to the solid phase by a cleavable linker, the oligomer synthesis was carried out, and the product then cleaved from the solid phase into solution. This method has the advantage that reactions may be forced to completion by the use of large excesses of reagents which may then be removed simply by washing. At the same time any soluble by-products are also removed. Many solid phases have been used, but the most important are derivatives of poly acrylic acid or polystyrene. These polymers are cross linked by inclusion of a divalent monomer to the extent necessary to give the required mechanical strength and solvent compatibility.
The development of combinatorial chemistry techniques (Gallup et al: J Med Chem, 37, 1233-1251 (1994)) wherein many thousands of compounds are synthesised in a single reaction by means of a resin mixing and splitting technique (Furka et al: Int J Pept Prot Res, 37, 487-493 (1991)) to give one (different) pure compound on each bead has lead to the expansion of solid phase chemistry from peptides and nucleotides to all types of organic chemistry, and this has required the development of new polymer supports with properties more suited to the different chemistries (Terrett et al: Tetrahedron 51, 8135-8173 (1995), Balkenhohl et al: Angew Chem Int Ed, 35, 2288-2337 (1996), Thompson and Ellman: Chem Rev, 96, 555-600 (1996)).
Resins such as chloromethyl or aminomethyl polystyrene have been modified to give linking groups with very special properties, making them suitable for SPOS. For example (4-hydroxymethylphenoxy)methyl polystyrene (the so-called xe2x80x98Wangxe2x80x99 resinxe2x80x94Wang: J Amer Chem Soc, 95, 1328-1333)) may be esterified to give esters which are stable to many chemical transformations, but which may be treated with, for example, trifluoroacetic acid to liberate the free acid.
The polymer support of the present invention can be similarly functionalised by converting the terminal hydroxy group into a cleavable linker. Cleavable linkers into which the hydroxy group may be converted are those known in the art for use in SPOS, especially those that can be esterified to give esters which is stable to many chemical transformations, but which may be treated, for example with either an acid, for example, trifluoroacetic acid, or a base or other nucleophile, depending upon the linker, to liberate a free acid or derivative thereof. For example the hydroxy group can be converted, usually via a better leaving group such as chloro, mesylate or tosylate, to a 4-hydroxymethylphenoxy or a 4-(4-methoxyphenyl(hydroxy)methyl)phenoxy group. The polymer supports can also be functionalised by conversion of the hydroxy group to an amino group, commonly via an intermediate leaving group such as chloro, mesylate or tosylate. The amino group can then be employed to attach a cleavable linker, for example via the formation of an amide moiety, thereby attaching the cleavable linker to the polyC2-4 alkyleneoxy chain. Cleavable linkers which can be attached to amino groups are well known in the art. Commonly, such linkers are derived from compounds comprising a free carboxylic acid moiety which reacts with the amino group to form an amide. Cleavable linkers which can be attached to amino groups include those derived from 4-hydroxymethylbenzoic acid and 4-hydroxymethylphenoxyacetic acid. For other examples of linkage agents which can be used to functionalise the terminal hydroxy group see Nova Biochem Combinatorial Chemistry Catalogue February 1997, pages 1-29, resins for SPOC.
Hence another aspect of the invention relates to a polymer support which comprises a cleavable linker or an amino group bonded to a cross-linked polymer through a polyC2-4 alkyleneoxy chain containing from 2 to 8 C2-4 alkyleneoxy groups
Accordingly, it will be recognised that the polymer supports according to the present invention can be represented by the general chemical formula:
[Polymer]-(OC2-4alkylene)2-8xe2x80x94X
wherein:
[Polymer] represents a cross-linked polymer; and
X represents OH; OP where P represents a protecting group; a cleavable linker; a leaving group; NH2; or NHY where Y represents a cleavable linker.
As indicated above, many of the polymer supports of the present invention, especially those derived from polymers with a lower degree of cross linking, swell in water or alcohols and at the same time do not have unduly slow relaxation times enabling NMR to be used. In addition, many of the polymer supports swell in methanol and ether, unlike polystyrene supports.
The invention will now be described, without limitation, by the following examples in which, unless otherwise stated:
a) FT-IR spectra were obtained using swollen gels in dichloromethane held between sodium chloride plates, and an ATI Genesis (Matteson)spectrometer.
b) 3C magic angle (MAS) NMR spectra were obtained using solvent swollen gels in the rotor of a Bruker MAS probe on a 400 MHz NMR spectrometer.
c) yields are given for illustration and are not necessarily the maximum attainable.
d) the following abreviations have been used: THF=tetrahydrofuran, DMF=N,N-dimethylformamide, FMOC=fluorenylmethoxycarbonyl, PEG=polyethyleneglycol and THP=tetrahydropyranyl.